Precision Biosystems Collaboration

Optimized Western Blot Assays Using BlotCycler™ Automated Wash System

Immunodetection of proteins is often performed manually and involves multiple steps that easily introduce bias and errors. We have compared the traditional manual Western blotting and a novel type of automatic Western blotting by using BlotCycler™ processor developed by Precision Biosystems, and DyLight™ fluorochrome conjugated secondary antibodies developed by Rockland Immunochemicals. The results of manual and automatic processing were comparable at RT, but automatic processing yielded consistently higher signal at 4ºC. Automatic processing using BlotCycler™ showed excellent reproducibility and can be efficiently used for optimization of primary and secondary antibodies concentration.

Summary

Rockland Immunochemicals and Precision Biosystems have used fluorescent labeled secondary antibodies to compare manual and automated processing. The multi-color visualization presented on the poster demonstrates that BlotCycler™ is able to produce clear and high quality results, allowing a straightforward process for the identification of individual protein.

Using two identical protocols, we have showed that BlotCycler™ increases the signal to noise ratio by reducing background and increasing signal intensity. BlotCycler™ provides extremely good reproducibility that is probably related to minimal variability in washing and incubation times. BlotCycler™ can be used to optimize the concentrations of primary and secondary antibodies potentially providing huge saving by decreasing the amount of antibody used.

Thus BlotCycler™ offers a time-saving alternative to manual processing of fluorescent western blot by providing very low background staining, high signal-to-noise ratio, ability to multiplex detection and excellent reproducibility. BlotCycler™ provides opportunity for easy optimization of western blotting and significantly improves the quality of the procedure.